Fluorescence in situ detection of short DNA sequences

描述(由申请人提供)：提出了一种全新的DNA荧光原位检测方法，使在非变性条件下在中期染色体铺展和间期核中检测短(约20个碱基对)单拷贝DNA序列成为可能。即将开发的短序列荧光原位检测方法(FISDOSS)将非常特异，因为环状探针将通过将合成的寡核苷酸连接到由特殊设计的肽核酸(PNA)打开的短DNA序列来组装。一种有效的污染免疫等温信号放大方法将提供高灵敏度：结合大量荧光标记的核苷酸对组装的环形探针进行滚环扩增(RCA)。所有程序将直接在玻片上进行，间期核和中期染色体的最终检测将通过使用荧光显微镜的标准技术完成。在第一阶段，原则证明实验将在任意选择的人类基因组独有的位置上进行。第一阶段的目标是证明，经过初步优化，短的特定序列可以有效和特异地在未变性的中期人类染色体中检测到。该方法将扩展到对人类基因组中各种独特位点的并行多重检测。为了证明FISDOSS适用于检测癌症的遗传标记，将测试与慢性淋巴细胞白血病(CLL)相关的12个合适的位置。该项目的实施将产生一种方便的荧光原位技术，在DNA水平上对癌症进行可靠和高灵敏的诊断具有巨大的潜力。

DESCRIPTION (provided by applicant): A radically new approach for the fluorescence in situ detection of DNA is proposed, which makes it possible to detect short (about 20-bp-long) single-copy DNA sequences in metaphase chromosome spreads and in interphase nuclei under non-denaturing conditions. The method of fluorescence in situ detection of short sequences (FISDOSS) to be developed will be exceedingly specific because a circular probe will be assembled via ligation of synthetic oligonucleotides on short DNA sequences opened up by specially designed peptide nucleic acids (PNAs). A high sensitivity will be provided by an efficient contamination-immune isothermal method of signal amplification: rolling-circle amplification (RCA) of the assembled circular probes with incorporation of numerous fluorescently labeled nucleotides. All procedures will be performed directly on slides and the final detection of interphase nuclei and metaphase chromosomes will be done by standard techniques using a fluorescent microscope. In Phase I, proof-of-principle experiments will be performed on arbitrarily chosen sites unique for the human genome. The goal of Phase I is to demonstrate, after initial optimization, that the short specific sequences can be effectively and specifically detected within non-denatured metaphase human chromosomes. The method will be extended to parallel multiple detection of various unique sites in the human genome. To demonstrate that FISDOSS is applicable to detect genetic markers of cancer, 12 appropriate sites associated with chronic lymphocytic leukemia (CLL) will be tested. The implementation of the project will yield a convenient fluorescent in situ technique with a great potential for reliable and highly sensitive diagnosis of cancer on the DNA level.